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Molecular and cellular neurosciences [journal]
- Calmodulin orchestrates the heteromeric assembly and the trafficking of KCNQ2/3 (Kv7.2/3) channels in neurons. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2013 Dec 11.
Mutations in KCNQ2 and KCNQ3 genes are responsible for benign familial neonatal seizures and epileptic encephalopathies. Some of these mutations have been shown to alter the binding of calmodulin (CaM) to specific C-terminal motifs of KCNQ subunits, known as the A and B helices. Here, we show that the mutation I342A in the A helix of KCNQ3 abolishes CaM interaction and strongly decreases the heteromeric association with KCNQ2. The assembly of KCNQ2 with KCNQ3 is essential for their expression at the axon initial segment (AIS). We find that the I342A mutation alters the targeting of KCNQ2/3 subunits at the AIS. However, the traffic of the mutant channels was rescued by provision of exogenous CaM. We show that CaM enhances the heteromeric association of KCNQ2/KCNQ3-I342A subunits by binding to their B helices in a calcium-dependent manner. To further assert the implication of CaM in channel assembly, we inserted a mutation in the second coil-coil domain of KCNQ2 (KCNQ2-L638P) to prevent its heteromerization with KCNQ3. We observe that the expression of a Ca(2+)-insensitive form of CaM favours the assembly of KCNQ3 with KCNQ2-L638P. Our data thus indicate that both apoCaM and Ca(2+)/CaM bind to the C-terminal domains of KCNQ2 and KCNQ3 subunits, and regulate their heteromeric assembly. Hence, CaM may control the composition and distribution of KCNQ channels in neurons.
- Gαz regulates BDNF-induction of axon growth in cortical neurons. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2013 Dec 7.
The disruption of neurotransmitter and neurotrophic factor signaling in the central nervous system (CNS) is implicated as the root cause of neuropsychiatric disorders, including schizophrenia, epilepsy, chronic pain, and depression. Therefore, identifying the underlying molecular mechanisms by which neurotransmitter and neurotrophic factor signaling regulates neuronal survival or growth may facilitate identification of more effective therapies for these disorders. Previously, our lab found that the heterotrimeric G protein, Gz, mediates crosstalk between G protein-coupled receptors and neurotrophin signaling in the neural cell line PC12. These data, combined with Gαz expression profiles - predominantly in neuronal cells with higher expression levels corresponding to developmental times of target tissue innervation - suggested that Gαz may play an important role in neurotrophin signaling and neuronal development. Here, we provide evidence in cortical neurons, both manipulated ex vivo and those cultured from Gz knockout mice, that Gαz is localized to axonal growth cones and plays a significant role in the development of axons of cortical neurons in the CNS. Our findings indicate that Gαz inhibits BDNF-stimulated axon growth in cortical neurons, establishing an endogenous role for Gαz in regulating neurotrophin signaling in the CNS.
- The Anaphase-Promoting Complex (APC) ubiquitin ligase regulates GABA transmission at the C. elegans neuromuscular junction. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2013 Dec 7.
Regulation of both excitatory and inhibitory synaptic transmission is critical for proper nervous system function. Aberrant synaptic signaling, including altered excitatory to inhibitory balance, is observed in numerous neurological diseases. The ubiquitin enzyme system controls the abundance of many synaptic proteins and thus plays a key role in regulating synaptic transmission. The Anaphase-Promoting Complex (APC) is a multi-subunit ubiquitin ligase that was originally discovered as a key regulator of protein turnover during the cell cycle. More recently, the APC has been shown to function in postmitotic neurons, where it regulates diverse processes such as synapse development and synaptic transmission at glutamatergic synapses. Here we report that the APC regulates synaptic GABA signaling by acting in motor neurons to control the balance of excitatory (acetylcholine) to inhibitory (GABA) transmission at the Caenorhabditis elegans neuromuscular junction (NMJ). Loss-of-function mutants in multiple APC subunits have increased muscle excitation at the NMJ; this phenotype is rescued by expression of the missing subunit in GABA neurons. Quantitative imaging and electrophysiological analyses indicate that APC mutants have decreased GABA release but normal cholinergic transmission. Consistent with this, APC mutants exhibit convulsions in a seizure assay sensitive to reductions in GABA signaling. Previous studies in other systems showed that the APC can negatively regulate the levels of the active zone protein SYD-2 Liprin-α. Similarly, we found that SYD-2 accumulates in APC mutants at GABAergic presynaptic sites. Finally, we found that the APC subunit EMB-27 CDC16 can localize to presynapses in GABA neurons. Together, our data suggest a model in which the APC acts at GABAergic presynapses to promote GABA release and inhibit muscle excitation. These findings are the first evidence for the APC in regulating transmission at inhibitory synapses and have implications for understanding nervous system pathologies, such as epilepsy, that are characterized by misregulated GABA signaling.
- Excitotoxic potential of exogenous ferritin and apoferritin: Changes in ambient level of glutamate and synaptic vesicle acidification in brain nerve terminals. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2013 Dec 6.
Ferritin, an iron storage protein, is present in the serum and cerebrospinal fluid, has receptors on the cell surface, able to penetrate the brain-blood barrier, can be secreted from the cells, and leaks from destroyed cell in insult and brain trauma. The effect of exogenous ferritin on the key characteristic of glutamatergic neurotransmission was assessed in rat brain nerve terminals (synaptosomes). Exogenous ferritin (80μg/ml, iron content 0.7 %) significantly increased the ambient level of L-[(14)C]glutamate (0.200±0.015 versus 0.368±0.016nmol/mg of protein) and endogenous glutamate (fluorimetric glutamate dehydrogenase assay) in the nerve terminals. This increase was not a result of augmentation of tonic release because the velocity of tonic release of L-[(14)C]glutamate was not changed significantly in ferritin-treated synaptosomes as compared to the control. Ferritin caused a decrease in synaptic vesicle acidification that was shown using fluorescent dye acridine orange. Iron-dependence of the effects of ferritin was analyzed with apoferritin (0.0025 % residual iron). Apoferritin weakly affected the proton electrochemical gradient of synaptic vesicles but increased the ambient level and decreased the initial velocity of uptake of L-[(14)C]glutamate by synaptosomes, nevertheless these effects were ~30 % lesser than those caused by ferritin. Exogenous ferritin can provoke the development of excitotoxicity increasing the ambient level of glutamate and lowering synaptic vesicle acidification and glutamate uptake in the nerve terminals, however these effects are not completely iron-dependent. Thus, in the CNS exogenous ferritin can act as modulator of glutamate homeostasis in iron-dependent and iron-independent manner.
- The schizophrenia susceptibility gene DTNBP1 modulates AMPAR synaptic transmission and plasticity in the hippocampus of juvenile DBA/2J mice. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2013 Dec 7.
The dystrobrevin binding protein (DTNBP) 1 gene has emerged over the last decade as a potential susceptibility locus for schizophrenia. While no causative mutations have been found, reduced expression of the encoded protein, dysbindin, was reported in patients. Dysbindin likely plays a role in the neuronal trafficking of proteins including receptors. One important pathway suspected to be affected in schizophrenia is the fast excitatory glutamatergic transmission mediated by AMPA receptors. Here, we investigated excitatory synaptic transmission and plasticity in hippocampal neurons from dysbindin-deficient sandy mice bred on the DBA/2J strain. In cultured neurons an enhancement of AMPAR responses was observed. The enhancement of AMPAR-mediated transmission was confirmed in hippocampal CA3-CA1 synapses, and was not associated with changes in the expression of GluA1-4 subunits or an increase in GluR2-lacking receptor complexes. Lastly, an enhancement in LTP was also found in these mice. These data provide compelling evidence that dysbindin, a widely suspected susceptibility protein in schizophrenia, is important for AMPAR-mediated synaptic transmission and plasticity in the developing hippocampus.
- Proteasome inhibition induces stress kinase dependent transport deficits - Implications for Alzheimer's disease. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2013 Nov 21.
Alzheimer's disease (AD) is characterized by accumulation of two misfolded and aggregated proteins, β-amyloid and hyperphosphorylated tau. Both cellular systems responsible for clearance of misfolded and aggregated proteins, the lysosomal and the proteasomal, have been shown to be malfunctioning in the aged brain and more so in patients with neurodegenerative diseases, including AD. This malfunction could be contributing to β-amyloid and tau accumulation, eventually aggregating in plaques and tangles. We have investigated the impact of decreased proteasome activity on tau phosphorylation as well as on microtubule stability and transport. To do this, we used our recently developed neuronal model where human SH-SY5Y cells obtain neuronal morphology and function through differentiation. We found that exposure to low doses of the proteasome inhibitor MG-115 caused tau phosphorylation, microtubule destabilization and disturbed neuritic transport. Furthermore, reduced proteasome activity activated several proteins implicated in tau phosphorylation and AD pathology, including c-Jun N-terminal kinase, c-Jun and extracellular signal-regulated protein kinase (ERK) 1/2. Restoration of the microtubule transport was achieved by inhibiting ERK 1/2 activation, and simultaneous inhibition of both ERK 1/2 and c-Jun reversed the proteasome inhibition-induced tau phosphorylation. Taken together, this study suggests that a decrease in proteasome activity can, through activation of c-Jun and ERK 1/2, result in several events related to neurodegenerative diseases. Restoration of proteasome activity or modulation of ERK 1/2 and c-Jun function can open new treatment possibilities against neurodegenerative diseases such as AD.
- The β2-adrenergic receptor as a surrogate odorant receptor in mouse olfactory sensory neurons. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2013 Nov 6.:1-10.
In the mouse, mature olfactory sensory neurons (OSNs) express one allele of one of the ~1200 odorant receptor (OR) genes, which encode G-protein coupled receptors (GPCRs). Axons of OSNs that express the same OR coalesce into homogeneous glomeruli at conserved positions in the olfactory bulb. ORs are involved in OR gene choice and OSN axonal wiring, but the mechanisms remain poorly understood. One approach is to substitute an OR genetically with another GPCR, and to determine in which aspects this GPCR can serve as a surrogate OR under experimental conditions. Here, we characterize a novel gene-targeted mouse strain in which the mouse β2-adrenergic receptor (β2AR) is coexpressed with tauGFP in OSNs that choose the OR locus M71 for expression (β2AR→M71-GFP). By crossing these mice with β2AR→M71-lacZ gene-targeted mice, we find that differentially tagged β2AR→M71 alleles are expressed monoallelically. The OR coding sequence is thus not required for monoallelic expression - the expression of one of the two alleles of a given OR gene in an OSN. We detect strong β2AR immunoreactivity in dendritic cilia of β2AR→M71-GFP OSNs. These OSNs respond to the β2AR agonist isoproterenol in a dose-dependent manner. Axons of β2AR→M71-GFP OSNs coalesce into homogeneous glomeruli, and β2AR immunoreactivity is detectable within these glomeruli. We do not find evidence for expression of endogenous β2AR in OSNs of wild-type mice, also not in M71-expressing OSNs, and we do not observe overt differences in the olfactory system of β2AR and β1AR knockout mice. Our findings corroborate the experimental value of the β2AR as a surrogate OR, including for the study of the mechanisms of monoallelic expression.
- Tropomyosins induce neuritogenesis and determine neurite branching patterns in B35 neuroblastoma cells. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2013 Nov 6.:11-21.
The actin cytoskeleton is critically involved in the regulation of neurite outgrowth.The actin cytoskeleton-associated protein tropomyosin induces neurite outgrowth in B35 neuroblastoma cells and regulates neurite branching in an isoform-dependent manner.Our data indicate that tropomyosins are key regulators of the actin cytoskeleton during neurite outgrowth.Revealing the molecular machinery that regulates the actin cytoskeleton during neurite outgrowth may provide new therapeutic strategies to promote neurite regeneration after nerve injury.The formation of a branched network of neurites between communicating neurons is required for all higher functions in the nervous system. The dynamics of the actin cytoskeleton is fundamental to morphological changes in cell shape and the establishment of these branched networks. The actin-associated proteins tropomyosins have previously been shown to impact on different aspects of neurite formation. Here we demonstrate that an increased expression of tropomyosins is sufficient to induce the formation of neurites in B35 neuroblastoma cells. Furthermore, our data highlight the functional diversity of different tropomyosin isoforms during neuritogenesis. Tropomyosins differentially impact on the expression levels of the actin filament bundling protein fascin and increase the formation of filopodia along the length of neurites. Our data suggest that tropomyosins are central regulators of actin filament populations which drive distinct aspects of neuronal morphogenesis.
- Dicer expression is essential for adult midbrain dopaminergic neuron maintenance and survival. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2013 Oct 31.:22-28.
The type III RNAse, Dicer, is responsible for the processing of microRNA (miRNA) precursors into functional miRNA molecules, non-coding RNAs that bind to and target messenger RNAs for repression. Dicer expression is essential for mouse midbrain development and dopaminergic (DAergic) neuron maintenance and survival during the early post-natal period. However, the role of Dicer in adult mouse DAergic neuron maintenance and survival is unknown. To bridge this gap in knowledge, we selectively knocked-down Dicer expression in individual DAergic midbrain areas, including the ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc) via viral-mediated expression of Cre in adult floxed Dicer knock-in mice (Dicer(flox/flox)). Bilateral Dicer loss in the VTA resulted in progressive hyperactivity that was significantly reduced by the dopamine agonist, amphetamine. In contrast, decreased Dicer expression in the SNpc did not affect locomotor activity but did induce motor-learning impairment on an accelerating rotarod. Knock-down of Dicer in both midbrain regions of adult Dicer(flox/flox) mice resulted in preferential, progressive loss of DAergic neurons likely explaining motor behavior phenotypes. In addition, knock-down of Dicer in midbrain areas triggered neuronal death via apoptosis. Together, these data indicate that Dicer expression and, as a consequence, miRNA function, are essential for DAergic neuronal maintenance and survival in adult midbrain DAergic neuron brain areas.
- Activation of brain endothelial cells by interleukin-1 is regulated by the extracellular matrix after acute brain injury. [Journal Article]
- Mol Cell Neurosci 2013 Nov.:93-103.
The extracellular matrix (ECM) of the central nervous system (CNS) is essential for normal brain function, whilst ECM remodelling is associated with cerebrovascular inflammation driven by the cytokine interleukin-1 (IL-1) after acute brain injury. The effect of ECM remodelling on endothelial activation during neuroinflammation remains unknown. Here we report that ECM remodelling in the cerebrovasculature critically regulates IL-1-induced endothelial cell activation after cerebral ischaemia; Expression levels of ECM molecules associated with the cerebrovasculature, namely fibronectin (FN) and collagen IV (Col IV), strongly increased in brain blood vessels after middle cerebral artery occlusion (MCAo) in a time-dependent manner, reaching a peak of vascular expression 48h after MCAo. In cultures, FN and Col IV (but also laminin-1 and fibrillin-1) promoted strong attachment of the GPNT endothelial cell line and primary rat brain endothelial cells, which was markedly inhibited by RGD (Arg-Gly-Asp) peptide, or specific integrin β1, α4, α5 and αv blockade. IL-1β-induced activation of extracellular-regulated kinase 1/2 (ERK1/2) and nuclear factor κB (NFκB), and synthesis of cytokine-induced neutrophil chemoattractant (CINC-1) were enhanced in cells plated onto ECM molecules, and these responses were inhibited by selective integrin blockade. Finally, increased ECM expression in vessels after MCAo was found associated with vinculin clustering, increased integrin β1 expression, and increased IL-1 receptor associated kinase-1 (IRAK-1) activity in endothelial cells and perivascular astrocytes. Therefore, our data indicate a novel function for the ECM in the regulation of cerebrovascular inflammation triggered by IL-1 during acute brain injury.